Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions

ABSTRACT

In some aspects, the invention relates to compositions comprising marrow infiltrating lymphocytes (“MILs”). The MILs may be activated MILs. In some aspects, the invention relates to methods for activating MILs, comprising incubating MILs in an environment comprising less than 21% oxygen. In some aspects, the invention relates to methods for treating cancer in a subject, comprising administering to the subject a composition comprising activated MILs.

PRIORITY CLAIM

This application is a Continuation of U.S. Serial application Ser. No.16/606,766, filed May 26, 2017, which is a Continuation of U.S. Serialapplication Ser. No. 15/112,353, filed on Jul. 18, 2016, which is U.S.national phase under U.S.C. § 371 of PCT application no:PCT/US15/048536, filed Sep. 4, 2015, which claims priority to U.S.Provisional Patent Application No. 62/045,782, filed on Sep. 4, 2014,and U.S. Provisional Patent Application No. 62/186,040, filed on Jun.29, 2015 The entire contents of these applications are incorporatedherein by reference in their entirety.

BACKGROUND

Myeloablative chemotherapy is an accepted therapy for many hematologicmalignancies including multiple myeloma albeit with minimal evidence oflong-term cures. However, the myeloablative therapy also provides anideal platform for the superimposition of immune-based therapies.Specifically, the lymphopenia resulting from high dose chemotherapyfacilitates homeostatic lymphocytic proliferation, eliminatestolerogenic antigen presenting cells (APCs), and induces cytokinerelease that generates a more favorable environment for adoptive T celltherapy. Indirect evidence that the immune system can contribute to theclinical benefits of high dose chemotherapy was shown with earlylymphoid recovery resulting in improved clinical outcomes in patientswith myeloma, lymphoma, and acute myeloid leukemia undergoing anautologous stem cell transplant. Furthermore, these improved outcomes inmyeloma correlated directly with the dose of autologous lymphocytesinfused from the apheresis product. Taken together, these data supportthe hypothesis that anti-tumor immunity can have clinically measurablebenefits and advances the question of how to harness such immunity toaugment the efficacy of currently available therapies.

The ability to eradicate measurable disease with adoptive T cell therapy(ACT) requires T cells to be appropriately activated and present insufficient numbers, possess appreciable anti-tumor activity, home to thetumor site, effectively kill the tumor upon encounter, and persist overtime. Stimulation of T cells with any technique including paramagneticbeads to which anti-CD3 and CD28 are bound can effectively reverse ananergic (tolerant) state, generate activated T cells, and significantlyexpand their numbers. While bead-bound anti-CD3 and CD28 provide astraightforward and robust T cell amplification in vitro, a majorlimitation of this approach is the non-specific stimulation of theentire T cell repertoire without enrichment of tumor specific T cells.One strategy to augment the tumor specificity of ACT is to use a T cellpopulation with greater endogenous tumor specificity. Such an enrichmentaccounts for the considerable anti-tumor activity of ACT using tumorinfiltrating lymphocytes (TILs) from metastatic melanoma. However, TILsare present only in a subset of patients with metastatic melanoma, andof those, successful TIL preparations can be achieved in only 60-70% ofpatients with harvestable tumor, which limits the general applicabilityof such an approach. Bone marrow is the tumor microenvironment for manyhematologic malignancies such as multiple myeloma, and thus,marrow-infiltrating lymphocytes (MILs) could be harnessed to generatetumor specific T cell therapy for these specific cancers. In contrast toTILs, MILs are present in all patients, can be obtained with a simplebed-side procedure, and can be rapidly expanded in all patients.

In hematologic malignancies, the bone marrow represents not only thesite of disease but also a unique microenvironment. Even in solidtumors, evidence exists that MILs can be enriched in memory oreffector-memory T cells. The immune component within the bone marrow isa reservoir of antigen experienced T cells for both tumor specific Tcells in host with early stage breast cancer as well as vaccine-primed Tcells. In the bone marrow, memory CD4 cells are maintained throughinteractions with IL-7 expressing stromal cells and CD8 cells aremaintained through the persistence of antigen expression and effectiveantigen presentation. As such, the heightened tumor specificity of MILsin this setting is likely due to the presence of tumor as a source ofantigen while their persistence is maintained through the unique immuneinteractions with stromal elements, cytokines, and antigen presentingcells capable of effective antigen presentation in this environment.

Ex-vivo activated MILs possess several essential properties for adoptiveT cell therapy. Upon activation, they demonstrate significant tumorspecificity compared to their peripheral blood lymphocyte counterparts,and they target a broad range of antigens present on both the maturemultiple myeloma plasma cells as well as their clonogenic precursors andeffectively kill multiple myeloma plasma cells. Similar to TILs, MILshave a greater endogenous polyclonal antigenic specificity thanperipheral lymphocytes. In contrast to TILs, MILs are present in allpatients and are obtained from a more immune responsivemicroenvironment. As such, MILs represent a novel and promisingtumor-specific approach to ACT for hematologic malignancies with bonemarrow involvement.

SUMMARY

In some aspects, the invention relates to a composition comprisingmarrow infiltrating lymphocytes (“MILs”). The composition may comprise apopulation of MILs that expresses CD3. For example, at least about 40%of the cells in the composition may be MILs from the population of MILsthat expresses CD3. For example, the composition may comprise MILs, and40% of the cells may express CD3 as determined by a flow cytometry gate;thus, at least about 40% of the cells in the composition would be fromthe population of MILs that expresses CD3. The composition may comprisea population of MILs that expresses interferon gamma (“IFNγ”). Forexample, at least about 2% of the cells in the composition may be MILsfrom the population of MILs that expresses IFNγ. The composition maycomprise a population of MILs that expresses CXCR4. For example, atleast about 98% of the cells in the composition may be MILs from thepopulation of MILs that expresses CXCR4. The composition may comprise apopulation of MILs that expresses CD4. The composition may comprise apopulation of MILs that expresses CD8. The composition may comprise apopulation of MILs that expresses 4-1BB. For example, at least about 21%of the cells in the composition may be MILs from the population of MILsthat expresses 4-1BB.

In some aspects, the invention relates to a method for activating marrowinfiltrating lymphocytes (“MILs”), comprising incubating MILs in anenvironment comprising less than 21% oxygen.

In some aspects, the invention relates to a method for treating cancerin a subject. The method may comprise administering to the subject acomposition comprising MILs. In some embodiments, the method comprisesremoving marrow infiltrating lymphocytes (“MILs”) from the subject; andincubating the MILs in an environment comprising less than 21% oxygen,thereby producing activated MILs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts flow cytometry results for MILs that were expanded undereither normoxic conditions or hypoxic conditions in media comprisinginterleukin 2 (+IL2) or media without interleukin 2 (No IL2). 35.09% ofthe gated cells grown under normoxic conditions in media comprisinginterleukin 2 were positive for CD3. 30.98% of the gated cells grownunder normoxic conditions in media lacking interleukin 2 were positivefor CD3. 89.01% of the gated cells grown under hypoxic conditions inmedia comprising interleukin 2 were positive for CD3. 89.96% of thegated cells grown under hypoxic conditions in media lacking interleukin2 were positive for CD3.

FIG. 2 is a chart showing the expansion of CD3⁺ cells for peripheralblood lymphocytes (PBLs) and MILs grown under normoxic or hypoxicconditions. Hypoxic conditions decreased the expansion of CD3⁺ PBLs butincreased the expansion of CD3⁺ MILs.

FIG. 3 is a graph comparing the expansion of CD3⁺ MILs after varioustime periods of incubation in either normoxic or hypoxic environments.Substantial expansion was observed after 7 days of incubation in ahypoxic environment compared to normoxia, and a CD3⁺ expansion peaked at11 days of incubation.

FIG. 4 is a graph depicting the tumor specificity of MILs expanded underhypoxic or normoxic conditions for either myeloma cell lines (U266/H929)or a control cell line (SW780), for cells that were expanded for either10 days (D10) or for 12 days (D12). Hypoxic cells were grown for 3 daysin an environment comprising 2% oxygen, followed by expansion in anormoxic environment (21% oxygen). On day 10, 4% of the cells expandedunder normoxic conditions were tumor specific as compared to 25.1% ofMILs expanded under hypoxic conditions as determined by the percent oftotal T cells that were CFSE low and producing interferon gamma (IFNγ)in response to tumor antigen.

FIG. 5 depicts flow cytometry results for tumor specificity of MILsexpanded under various conditions utilizing gates for CD3 and INFγ.After seven days of expansion, 18.26% of MILs grown under hypoxicconditions were positive for both CD3 and interferon gamma. In contrast,only 1.72% of MILs grown under normoxic conditions were positive forboth CD3 and interferon gamma.

FIG. 6 is a graph that depicts the in vivo expansion of lymphocytespost-autologous transplant including in subjects receiving MILs grownunder various conditions. The x-axis corresponds to the number of dayspost-transplant and the y-axis corresponds to the average absolutelymphocyte count per microliter for the subjects. The graph suggeststhat MILs grown under hypoxic conditions continue to expand in humansubjects more than MILs grown under only normoxic conditions.

FIG. 7 depicts flow cytometry results for MILs expanded under variousoxygen conditions utilizing gates for CXCR4 and either CD4 or CD8.28.38% of the total MILs population expanded under normoxic conditionswere both CXCR4 positive and CD4 positive, with a mean fluorescenceintensity of 3.9. 68.91% of MILs expanded under hypoxic conditions wereboth CXCR4 positive and CD4 positive, with a mean fluorescence intensityof 5.4. 8.02% of MILs expanded under normoxic conditions were both CXCR4positive and CD8 positive, with a mean fluorescence intensity of 4.6.11.08% of MILs expanded under hypoxic conditions were both CXCR4positive and CD8 positive. These results suggest that hypoxia increasesboth the number of cells expressing CXCR4 as well as degree ofexpression per cell (MFI), which increases the likelihood of these cellsto migrate to the bone marrow upon infusion.

FIG. 8 consists of three panels, labeled panels (A), (B), and (C). PanelA depicts flow cytometry results for peripheral blood lymphocytes (PBL)and MILs prior to cell expansion, utilizing gates for CD4, CD8, and4-1BB. Panel B depicts flow cytometry results for PBLs and MILsfollowing expansion under normoxic conditions, utilizing gates for CD4,CD8, and 4-1BB. As shown, 4-1BB expression in PBLs decreased in CD8PBLs, from 11.34% to 0.34%, and MILs CD8 showed a decrease from 15.1% to7.54%. Panel C depicts flow cytometry results for PBLs and MILsfollowing expansion under hypoxic conditions, utilizing gates for CD4,CD8, and 4-1BB. PBLs downregulated 4-1BB following expansion underhypoxic conditions (CD8 PBLs: baseline 11.34% to 0%), whereas MILsupregulated 4-1BB following expansion under hypoxic conditions (CD8MILs: baseline 15.1% to 21.79%).

FIG. 9 is a graph that shows that the growth of MILs under hypoxicconditions increases the number of CD3⁺ cells relative to growth underonly normoxic conditions.

FIG. 10 shows flow cytometry results indicating that the expansion ofMILs under hypoxic conditions results in a higher percentage ofCD4⁺/4-1BB⁺ cells than either expansion of PBLs under hypoxic conditionsor the expansion of MILs under normoxic conditions.

FIG. 11 is a graph that shows the ex vivo expansion of MILs. MILsexpanded under hypoxic conditions resulted in a larger dose relative toMILs expanded under only normoxic conditions.

DETAILED DESCRIPTION

A major objective to achieve with adoptive T cell therapy is the abilityto grow the largest number of tumor specific T cells that willsubsequently also expand in vivo upon reinfusion and persist over time.In some aspects, the invention relates to a novel approach to T cellexpansion that takes advantage of intrinsic properties of marrowinfiltrating lymphocytes (“MILs”). Specifically, MILs significantlydiffer from peripheral lymphocytes (PBLs). For example, MILs are moreeasily expanded, upregulate activation markers to a greater extent thanPBLs, maintain more of a skewed Vβ repertoire, traffic to the bonemarrow, and most importantly, possess significantly greater tumorspecificity. MILs anti-myeloma immunity correlates directly withclinical response; however, no in vivo T cell expansion or persistentclinical response has previously been observed following infusion.

Culturing MILs at an O₂ level of less than 21% oxygen, such as about 1%oxygen to about 7% oxygen or about 1% oxygen to about 3% oxygen, e.g.,2% oxygen (hypoxic conditions) increases both the overall expansion ofthe cells as well as their ability to recognize tumor cells relative toculturing in only normoxic conditions. Thus, in some embodiments, theinvention relates to a method for the preparation of MILs fortherapeutic use comprising one or more of the following. Bone marrow maybe collected from a patient. The collected bone marrow may be frozen orimmediately used, for example, to create tumor specific MILs. If thebone marrow is frozen, it is preferably thawed before incubation. Thebone marrow may be treated to purify MILs through methods known to oneof ordinary skill in the art. The MILs may be activated, for example,with beads, e.g., anti-CD3/CD28 beads. The ratio of beads to cells inthe solution may vary; in some preferred embodiments, the ratio is 3to 1. Similarly, the MILs may be expanded in the presence of one or moreantibodies, antigens, and/or cytokines, e.g., in the absence ofanti-CD3/CD28 beads. The cell count for the collected bone marrow may bedetermined, for example, to adjust the amount of beads, antibodies,antigens, and/or cytokines to be added to the MILs. In some embodiments,MILs are captured using beads specifically designed to collect thecells.

The collected MILs are preferentially grown in a hypoxic environment,e.g., for a first period of time. In some embodiments, MILs may beplaced in a tissue culture bag in X-VIVO™ 15 media supplemented with 2%AB serum and 200 U of IL2. It is contemplated that other cultureconditions and elements may be used as recognized by a person ofordinary skill in the art. The MILs may be grown in an environment ofabout 1% to about 7% oxygen (hypoxia; hypoxic conditions), preferablyabout 1% to about 3% oxygen, such as about 2% oxygen, for about 3 toabout 20 days (i.e., a first period of time), such as about 3 to about10 days, such as 4 days. The hypoxic environment may be created, forexample, by adding nitrous oxide to the container in which the cells aregrown. In some embodiments, the hypoxic environment may be created byutilizing a hypobaric chamber. After the hypoxic growth, the MILs may begrown in a normoxic environment, e.g., 21% oxygen. In some preferredembodiments, the MILs are grown in normoxic conditions for an additionalabout 3 to about 7 days (i.e., a second period of time), e.g., for atotal of about 3 to about 27 days of growth, such as about 3 to about 10days growth. The grown cells may then be either administered to apatient (e.g., either the patient or an allogenic recipient) or storedfor future use.

MILs collected from the patient's bone marrow and treated in accordancewith a method described herein, namely, under hypoxic conditions for afirst period of time followed by normoxic conditions for a second periodof time, perform unexpectedly better than peripheral blood lymphocytes(PBLs) subjected to the same procedure. The enhanced abilities of theMILs, as shown below, include marked expansion both in vitro and invivo, enhanced expression of biological markers such as 4-1BB, andincreased tumor specificity.

A person of ordinary skill in the art would recognize that the procedureof the present invention can be utilized to treat many different typesof cancer, including, myeloma, lung cancer, and breast cancer. As bonemarrow is a reservoir of central memory cells, tumor specific T cellsfrom any variety of cancers have been found in the bone marrow ofpatients. As disclosed herein, hypoxic culturing conditions increaseboth expansion as well as tumor specificity, and thus, this approach maybe used to grow MILs from a wide range of cancer patients. In somepreferred embodiments, a patient's MILs are collected, expanded inhypoxic conditions for a first period of time, e.g., for about 1 day toabout 20 days, and then expanded in normoxic conditions for a secondperiod of time, e.g., about 3 days to about 7 days. The cells may thenbe provided to the patient for treatment or stored for future use.

In some aspects, the invention relates to the finding that expandingMILs in a hypoxic environment allows for in vivo T cell expansionfollowing infusion. Specifically, MILs were grown in 2% O₂ (hypoxia) for3 days followed by a switch to 21% O₂ (normoxia), resulting in almost a10-fold greater tumor specificity (FIG. 4). Taken together, these datasuggest that such growth conditions are capable of increasing theabsolute number of tumor specific MILs obtained from the same sourcerelative to MILs that a grown under only normoxic conditions.

All experiments were performed using MILs products from patient samples.As shown in FIG. 3, expansion of a full scale clinical MILs product inhypoxic conditions dramatically increased T cell numbers. It should benoted that with normoxic conditions, it was difficult to expand MILspast 7 days. In this experiment, the day-7 expansion was 36.3 fold innormoxia versus 119 fold in hypoxia. Furthermore, the cells grown underhypoxic conditions continued to expand up to 12 days and reached a total220-fold expansion at 11 days.

In addition to obtaining better expansions and greater tumorspecificity, growth conditions were optimized to maximize T cellsurvival. Expression of 4-1BB has been shown to be a key regulator ofmany of these properties. It can regulate T cell expansion, reduceapoptosis, augment the cytotoxic activity of CD8 cells, and enhancesurvival. Taken together, 4-1BB expression on activated MILs may be animportant regulator of heightened survival and tumor specificity. Thus4-1BB expression was examined on MILs and compared to that on PBLs grownin various conditions. As shown in FIG. 8, the baseline expression of4-1BB was greater in MILs than PBLs (18.2% v 8.1%). Interestingly, Tcell expansion in normoxia reduced its expression in both populations(MILs 10.7%, PBLs 2.8%) whereas expansion in hypoxia significantlyincrease 4-1BB expression in MILs (43.4%) and completely abrogated itsexpression in PBLs (0%). These data again underscore the significantdifferences between PBLs and MIL also demonstrate that 4-1BBupregulation depends upon more factors than simply hypoxic growthconditions.

These culture conditions have been adopted to a clinical trial, andhypoxic growth conditions increased the total T cell expansion from anaverage of 7.9E9 to 1.8E10. Furthermore, hypoxic growth conditions alsoenabled the observation of in vivo T cell expansion (FIG. 6, whichdepicts the total lymphocyte counts through day 60 for all patients).

In some aspects, the invention relates to a composition comprisingmarrow infiltrating lymphocytes (“MILs”). The MILs may be activatedMILs.

In preferred embodiments, the composition comprises a population of MILsthat expresses CD3, i.e., wherein each cell in the population of MILsthat expresses CD3 is a marrow infiltrating lymphocyte that expressesCD3, e.g., as detected by flow cytometry. For example, at least about40% of the cells in the composition may be MILs from the population ofMILs that express CD3, such as at least about 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, or even at least about 89% of thecells in the composition. In one preferred embodiment, at least about80% of the cells in the composition may be MILs from the population ofMILs that express CD3. In some embodiments, about 40% to about 100% ofthe cells in the composition may be MILs from the population of MILsthat express CD3, such as about 45% to about 100%, about 50% to about100%, about 55% to about 100%, about 60% to about 100%, about 65% toabout 100%, about 70% to about 100%, about 75% to about 100%, about 80%to about 100%, about 85% to about 100%, about 86% to about 100%, about87% to about 100%, about 88% to about 100%, or even about 89% to about100% of the cells in the composition. In some embodiments, thecomposition comprises either a population of MILs that do not expressCD3, e.g., as detected by flow cytometry, or a population of MILs thatexpresses low levels of CD3, i.e., relative to the expression level ofMILs from the population of MILs that express CD3.

In some embodiments, the composition comprises a population of MILs thatexpresses interferon gamma (“IFNγ”), i.e., wherein each cell in thepopulation of MILs that expresses IFNγ is a marrow infiltratinglymphocyte that expresses IFNγ, e.g., as detected by flow cytometry. Forexample, at least about 2% of the cells in the composition may be MILsfrom the population of MILs that express IFNγ, such as at least about2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,or even at least about 18% of the cells in the composition. In someembodiments, about 2% to about 100% of the cells in the composition maybe MILs from the population of MILs that express IFNγ, such as about 2%to about 100%, about 3% to about 100%, about 4% to about 100%, about 5%to about 100%, about 6% to about 100%, about 7% to about 100%, about 8%to about 100%, about 9% to about 100%, about 10% to about 100%, about11% to about 100%, about 12% to about 100%, about 13% to about 100%,about 14% to about 100%, about 15% to about 100%, about 16% to about100%, about 17% to about 100%, or even about 18% to about 100% of thecells in the composition. In some embodiments, the composition compriseseither a population of MILs that do not express IFNγ, e.g., as detectedby flow cytometry, or a population of MILs that expresses low levels ofIFNγ, i.e., relative to the expression level of MILs from the populationof MILs that express IFNγ.

In some embodiments, the composition comprises a population of MILs thatexpresses CXCR4, i.e., wherein each cell in the population of MILs thatexpresses CXCR4 is a marrow infiltrating lymphocyte that expressesCXCR4, e.g., as detected by flow cytometry. For example, at least about98% of the cells in the composition may be MILs from the population ofMILs that express CXCR4, such as at least about 98.1%, 98.2%, 98.3%,98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, or even at least about 99.7% of the cells in thecomposition. In some embodiments, about 98% to about 100% of the cellsin the composition may be MILs from the population of MILs that expressCXCR4, such as at least about 98.1% to about 100%, about 98.2% to about100%, about 98.3% to about 100%, about 98.4% to about 100%, about 98.5%to about 100%, about 98.6% to about 100%, about 98.7% to about 100%,about 98.8% to about 100%, about 98.9% to about 100%, about 99.0% toabout 100%, about 99.1% to about 100%, about 99.2% to about 100%, about99.3% to about 100%, about 99.4% to about 100%, about 99.5% to about100%, about 99.6% to about 100%, or even about 99.7% to about 100% ofthe cells in the composition. In some embodiments, the compositioncomprises either a population of MILs that do not express CXCR4, e.g.,as detected by flow cytometry, or a population of MILs that expresseslow levels of CXCR4, i.e., relative to the expression level of MILs fromthe population of MILs that express CXCR4.

In some embodiments, the composition comprises a population of MILs thatexpresses CD4. The population of MILs that expresses CD4 may comprise aplurality of MILs that expresses CXCR4.

The population of MILs that expresses CD4 may comprise a plurality ofMILs that expresses 4-1BB. For example, at least about 21% of the cellsin the composition may be MILs from the plurality of MILs that expresses4-1BB, such as at least about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, or evenat least about 43% of the cells in the composition. In some embodiments,about 21% to about 100% of the cells in the composition may be MILs fromthe plurality of MILs that expresses 4-1BB, such as about 22% to about100%, about 23% to about 100%, about 24% to about 100%, about 25% toabout 100%, about 26% to about 100%, about 27% to about 100%, about 28%to about 100%, about 29% to about 100%, about 30% to about 100%, about31% to about 100%, about 32% to about 100%, about 33% to about 100%,about 34% to about 100%, about 35% to about 100%, about 36% to about100%, about 3′7% to about 100%, about 38% to about 100%, about 39% toabout 100%, about 40% to about 100%, about 41% to about 100%, about 42%to about 100%, or even about 43% to about 100% of the cells in thecomposition.

The composition may comprise a population of MILs that expresses CD8.The population of MILs that expresses CD8 may comprise a plurality ofMILs that expresses CXCR4.

The population of MILs that expresses CD8 may comprise a plurality ofMILs that expresses 4-1BB. For example, at least about 21% of the cellsin the composition may be MILs from the plurality of MILs that expresses4-1BB, such as at least about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20% or even at least about 21% of the cells in thecomposition. In some embodiments, about 2% to about 100% of the cells inthe composition may be MILs from the plurality of MILs that expresses4-1BB, such as about 8% to about 100%, about 9% to about 100%, about 10%to about 100%, about 11% to about 100%, about 12% to about 100%, about13% to about 100%, about 14% to about 100%, about 15% to about 100%,about 16% to about 100%, about 17% to about 100%, about 18% to about100%, about 19% to about 100%, about 20% to about 100%, or even about21% to about 100% of the cells in the composition.

In some embodiments, the composition comprises a population of MILs thatexpresses 4-1BB. For example, at least about 21% of the cells in thecomposition may be MILs from the population of MILs that expresses4-1BB, such as at least about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, or evenat least about 43% of the cells in the composition. In some embodiments,about 21% to 100% of the cells in the composition may be MILs from thepopulation of MILs that expresses 4-1BB, such as about 22% to about100%, about 23% to about 100%, about 24% to about 100%, about 25% toabout 100%, about 26% to about 100%, about 27% to about 100%, about 28%to about 100%, about 29% to about 100%, about 30% to about 100%, about31% to about 100%, about 32% to about 100%, about 33% to about 100%,about 34% to about 100%, about 35% to about 100%, about 36% to about100%, about 37% to about 100%, about 38% to about 100%, about 39% toabout 100%, about 40% to about 100%, about 41% to about 100%, about 42%to about 100%, or even about 43% to about 100% of the cells in thecomposition. In some embodiments, the composition comprises either apopulation of MILs that do not express 4-1BB, e.g., as detected by flowcytometry, or a population of MILs that expresses low levels of 4-1BB,i.e., relative to the expression level of MILs from the population ofMILs that express 4-1BB.

In some aspects, the invention relates to a method for preventing ortreating cancer in a subject, comprising administering to the subjectany one of the compositions described herein. In preferred embodiments,the method comprises administering to the subject atherapeutically-effective amount of any one of the compositionsdescribed herein. In preferred embodiments, the method comprisesadministering to the subject a therapeutically-effective amount of MILs,e.g., activated MILs, as described herein. The subject may have aneoplasm, such as cancer. For example, the subject may have multiplemyeloma. The subject may be a human subject.

In some aspects, the invention relates to a method for making acomposition as described herein, comprising incubating MILs in a hypoxicenvironment. In some aspects, the invention relates to a method foractivating marrow infiltrating lymphocytes (“MILs”), comprisingincubating MILs in a hypoxic environment.

In some aspects, the invention relates to a method for method fortreating cancer in a subject. The method may comprise removing marrowinfiltrating lymphocytes (“MILs”) from the subject; incubating the MILsin a hypoxic environment, thereby producing activated MILs; andadministering the activated MILs to the subject.

The hypoxic environment may comprise less than about 21% oxygen, such asless than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, or less than about 3% oxygen. For example, thehypoxic environment may comprise about 0% oxygen to about 20% oxygen,such as about 0% oxygen to about 19% oxygen, about 0% oxygen to about18% oxygen, about 0% oxygen to about 17% oxygen, about 0% oxygen toabout 16% oxygen, about 0% oxygen to about 15% oxygen, about 0% oxygento about 14% oxygen, about 0% oxygen to about 13% oxygen, about 0%oxygen to about 12% oxygen, about 0% oxygen to about 11% oxygen, about0% oxygen to about 10% oxygen, about 0% oxygen to about 9% oxygen, about0% oxygen to about 8% oxygen, about 0% oxygen to about 7% oxygen, about0% oxygen to about 6% oxygen, about 0% oxygen to about 5% oxygen, about0% oxygen to about 4% oxygen, or about 0% oxygen to about 3% oxygen. Inpreferred embodiments, the hypoxic environment comprises about 1% toabout 7% oxygen. The hypoxic environment may comprise about 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, or about 0% oxygen. In preferred embodiments, the hypoxicenvironment comprises about 7%, 6%, 5%, 4%, 3%, 2%, or 1% oxygen.

Incubating MILs in a hypoxic environment may comprise incubating theMILs, e.g., in tissue culture medium, for at least about 1 hour, such asat least about 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42hours, 48 hours, 60 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or even at least about14 days. Incubating may comprise incubating the MILs for about 1 hour toabout 30 days, such as about 1 day to about 20 days, about 1 day toabout 14 days, or about 1 day to about 12 days. In some preferredembodiments, incubating MILs in a hypoxic environment comprisesincubating the MILs in a hypoxic environment for about 2 days to about 5days. The method may comprise incubating MILs in a hypoxic environmentfor about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 day,9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In somepreferred embodiments, the method comprises incubating the MILs in ahypoxic environment for about 3 days.

In preferred embodiments, the method further comprises incubating theMILs in a normoxic environment, e.g., after incubating the MILs in ahypoxic environment.

The normoxic environment may comprise at least about 21% oxygen. Thenormoxic environment may comprise about 5% oxygen to about 30% oxygen,such as about 10% oxygen to about 30% oxygen, about 15% oxygen to about25% oxygen, about 18% oxygen to about 24% oxygen, about 19% oxygen toabout 23% oxygen, or about 20% oxygen to about 22% oxygen. In someembodiments, the normoxic environment comprises about 21% oxygen.

Incubating MILs in a normoxic environment may comprise incubating theMILs, e.g., in tissue culture medium, for at least about 1 hour, such asat least about 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42hours, 48 hours, 60 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or even at least about14 days. Incubating may comprise incubating the MILs for about 1 hour toabout 30 days, such as about 1 day to about 20 days, about 1 day toabout 14 days, about 1 day to about 12 days, or about 2 days to about 12days.

EXEMPLIFICATION Example 1. Activation and Expansion of T Cells inHypoxic and Normoxic Environments

Bone marrow (BM) T cell numbers are determined using flow cytometry.Anti-CD 3/anti-CD28 beads are added at the pre-determined ratio(beads:CD3 cell) in media with recombinant human cytokines at apredetermined concentration. Cells are plated in a plate, flask, or bag.Hypoxic conditions are achieved by flushing either the hypoxic chamberor cell culture bag for 3 minutes with a 95% Nitrogen and 5% CO₂ gasmixture. The receptacle is then filled with this gas mixture for 30seconds. This leads to a 2% or less O₂ gas in the receptacle. Cells arecultured at 37 C for 3 or more days and the hypoxic air is released andreplaced with normoxic (21% atmospheric oxygen) levels.

Example 2. Phenotypic Determination of Cell Types

Cells are stained with flurochrome conjugated antibodies for the desireddetermination. CD3, CD4, CD8, CXCR4, 41BB, CD27, CD28, CTLA-4, PD-1,CD45RO, CD62L, CD95, IFNg, IL17, live/dead dye, and/or other antibodiesof interest that are directly conjugated to flurochromes are used withappropriate isotype controls. Briefly, 1×10⁶ cells or less are washedwith FACS buffer (1×HBSS/2% FBS/0.5% EDTA/0.5% NaAzide) or similar washbuffer in either a plate or a tube by spinning in a centrifuge. The washbuffer is removed and antibodies and isotype controls are added atpredetermined concentrations. The cells are stained between 7-30 minutesat either room temperature or at 4° C. The cells are washed 2× with washbuffer and re-suspended in minimal wash buffer. The cells are then runon a flow cytometer that has been properly compensated and prepared forthe flurochromes that are being utilized. 10,000 or greater numbers ofevents are collected for each sample. Data is analyzed utilizing FACSanalysis software. Flurochrome labelled cells are compared to isotypecontrols for back ground removal. Data is graphed as % positive-%background.

Example 3. Determination of Fold Expansion

Bone marrow cells are enumerated at the beginning of expansion. Thepercentage of CD3+ cells is determined utilizing flow cytometry. Thetotal number of CD3+ MILs is determined by multiplying the total numberof cells with the percentage of CD3=total number of CD3+ MILs inculture. On the final day of culture the cells are harvested and counted(both manually and with an automated cell counter). The percentage ofCD3+ is determined. The total number of CD3+ cells on the final day ofculture is determined by multiplying total cell number with thepercentage of CD+=total number of CD3+ MILs harvested. Total foldexpansion=Total number of CD3+ MILs harvested on the final day ofculture divided by the total number of CD3+ MILs on the initial day ofculture.

Example 4. Tumor Specificity

MILs are labelled with CFSE or a similar cell membrane integration dyeaccording to the manufacturers' protocol. Autologous BM is pulsed witheither media alone, a negative control (unrelated protein or lysate) orwith the protein or lysate of interest. CFSE labelled cells are thenco-cultured with pulsed autologous BM for 2-7 days. Cells are harvestedfrom the tissue culture plate or flask and then stained with CD3extracellularly and intracellularly with IFNg. Analysis of tumorspecificity is determined by gating on CD3+ cells that are CFSE low(divided cells) and that are producing IFNg.

Example 5. Activation and Expansion of T Cells in Hypoxic and NormoxicEnvironments

MILs were grown in 2% O₂ (hypoxia) for 3 days followed by a switch to21% O₂ (normoxia) for an additional 5 days in the presence or absence ofIL-2. As shown in FIG. 9, growth in hypoxia followed by normoxiaresulted in almost a 10-fold increase in expansion as compared to MILsgrown exclusively in normoxic conditions. Tumor specificity was alsomarkedly enhanced as shown on FIG. 4. On day 10, 4% of CFSE-low cellswere tumor specific in normoxic conditions as opposed to 25.1% for MILsgrown in hypoxic conditions. Taken together, these data suggest thatthese growth conditions are capable of increasing the absolute number oftumor specific upon activation.

The experiments in the preceding paragraph were performed on a smallsample. MILs product from a patient in a first clinical study was alsoexpanded using this method. As shown in FIG. 3, expansion of MILs inthese conditions dramatically increased T cell numbers. Growth innormoxic conditions was seldom capable of expanding MILs past 7 days. Inthis experiment, the day-7 fold expansion was 36.3 fold in normoxicconditions as oppose to 119 in hypoxic conditions. Furthermore, thecells continued to expand up to 12 days and reached a total 220-foldexpansion at 11 days before beginning to die.

Expression of 4-1BB has been shown to be a key regulator of many ofthese properties. It can regulate T cell expansion, reduce apoptosis,augment the cytotoxic activity of CD8 cells and enhances survival.Furthermore, HIF1α regulates survival of antigen-driven T cells.Chimeric antigen receptor (CAR) modified T cells with the vectorexpressing 4-1BB have shown significant in vivo expansion. Takentogether, 4-1BB expression on activated MILs can be an importantregulator of heightened survival and tumor specificity. One markedadvantage of the method disclosed herein is that there is no need tomodify the MILs in order to achieve 4-1BB enhanced expression. Thisadvantage is shown in FIG. 10 where MILs or PBLs were grown in eithernormoxic or hypoxic conditions. Baseline expression of 4-1BB wasevaluated in MILs and compared to PBLs. As shown, 4-1BB expression wasgreater in MILs than PBLs (18.2% v 8.1%). Interestingly, T cellexpansion in normoxia reduced its expression in both populations (MILs10.7%, PBLs 2.8%) whereas expansion in hypoxia significantly increases4-1BB expression in MILs (43.4%) and completely abrogated its expressionin PBLs (0%). These data again underscore the significant differencesbetween PBLs and MIL also demonstrates that 4-1BB upregulation dependsupon more factors than simply hypoxic growth conditions. Moreimportantly, the unexpected upregulation of 4-1BB demonstrates that themethod of the present invention makes a significant difference in thetreatment of patients using hypoxically grown MILs. Similar results wereobserved with CD8 cells (FIG. 8).

FIG. 11 shows the results of dosing for clinical treatment. In J0770,MILs were grown in static cultures in normoxic conditions. J0997—MILswere grown in the WAVE in normoxic conditions. J1343—MILs were grown for3 days in hypoxic conditions followed by normoxic conditions. In FIG. 6the absolute lymphocyte counts are graphed for the 3 trialspost-autologous stem cell transplant. J1343 is a randomized trial inwhich patients either received hypoxic MILs or had no MILs infusedpost-transplant.

As shown in FIG. 11, the growth conditions of the present method haveincreased total T cell expansion from an average of 7.9E9 to 1.8E10.Furthermore, it shows for the first time in vivo T cell expansion asshown in FIG. 6, which is directly related to the efficacy of the methodin treatment of patients. Depicted in the graph are the total lymphocytecounts through day 60 for a first set of patients.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A method for treating a subject having cancer withtherapeutic activated marrow infiltrating lymphocytes, the methodcomprising the steps of: (a) culturing a bone marrow sample obtainedfrom the subject having cancer with an anti-CD3 antibody and ananti-CD28 antibody in a hypoxic environment to produce hypoxic-activatedmarrow infiltrating lymphocytes; (b) culturing the hypoxic-activatedmarrow infiltrating lymphocytes in a normoxic environment in thepresence of IL-2 to produce the therapeutic activated marrowinfiltrating lymphocytes; and (c) administering the therapeuticactivated marrow infiltrating lymphocytes to the subject having cancer.2. The method of claim 1, wherein said hypoxic environment comprisesless than about 21% oxygen.
 3. The method of claim 1, wherein saidhypoxic environment comprises about 0% oxygen to about 20% oxygen. 4.The method of claim 1, wherein said hypoxic environment comprises about0% oxygen to about 10% oxygen.
 5. The method of claim 1, wherein saidhypoxic environment comprises about 0% oxygen to about 3% oxygen.
 6. Themethod of claim 1, wherein said hypoxic environment comprises less than2% oxygen.
 7. The method of claim 1, wherein the hypoxic environmentcomprises between about 1% and about 2% oxygen.
 8. The method of claim1, wherein the bone marrow sample is cultured in the hypoxic environmentfor about 24 hours.
 9. The method of claim 1, wherein the bone marrowsample is cultured in the hypoxic environment for about 2 days.
 10. Themethod of claim 1, wherein the bone marrow sample is cultured in thehypoxic environment for about 3 days.
 11. The method of claim 1, whereinthe bone marrow sample is cultured in the hypoxic environment for about2 to about 5 days.
 12. The method of claim 1, wherein thehypoxic-activated marrow infiltrating lymphocytes are cultured in thenormoxic environment for about 2 to about 12 days.
 13. The method ofclaim 1, wherein the hypoxic-activated marrow infiltrating lymphocytesare cultured in the normoxic environment for about 6 days.
 14. Themethod of claim 1, wherein the hypoxic-activated marrow infiltratinglymphocytes are cultured in the normoxic environment for about 9 days.15. The method of claim 1, further comprising the step of: removing abone marrow sample from said subject having cancer prior to step (a).16. The method of claim 1, wherein the anti-CD3 antibody and theanti-CD28 antibody are bound on a bead.
 17. The method of claim 1,wherein the cancer is a hematological cancer.
 18. The method of claim 1,wherein the cancer is a solid tumor.
 19. The method of claim 1, whereinthe cancer is lung cancer.
 20. The method of claim 1, wherein the canceris breast cancer.
 21. The method of claim 1, wherein said normoxicenvironment comprises between about 21% oxygen and about 30% oxygen. 22.The method of claim 1, wherein said normoxic environment comprisesbetween about 21% oxygen and about 25% oxygen.
 23. The method of claim1, wherein said normoxic environment comprises between about 21% oxygenand about 24% oxygen.
 24. The method of claim 1, wherein said normoxicenvironment comprises between about 21% oxygen and about 23% oxygen. 25.A method for treating a subject having cancer with therapeutic activatedmarrow infiltrating lymphocytes, the method comprising the steps of: (a)culturing a bone marrow sample obtained from the subject having cancerwith anti-CD3/anti-CD28 beads in a hypoxic environment for about 2 daysto about 5 days to produce hypoxic-activated marrow infiltratinglymphocytes; (b) culturing the hypoxic-activated marrow infiltratinglymphocytes in a normoxic environment of at least about 21% oxygen forabout 2 to about 12 days in the presence of IL-2 to produce thetherapeutic activated marrow infiltrating lymphocytes; and (c)administering the therapeutic activated marrow infiltrating lymphocytesto the subject having cancer.
 26. The method of claim 25, wherein saidhypoxic environment comprises less than about 21% oxygen.
 27. The methodof claim 25, wherein said hypoxic environment comprises about 0% oxygento about 20% oxygen.
 28. The method of claim 25, wherein said hypoxicenvironment comprises about 0% oxygen to about 10% oxygen.
 29. Themethod of claim 25, wherein said hypoxic environment comprises about 0%oxygen to about 3% oxygen.
 30. The method of claim 25, wherein saidhypoxic environment comprises less than 2% oxygen.
 31. The method ofclaim 25, wherein the hypoxic environment comprises between about 1% andabout 2% oxygen.
 32. The method of claim 25, wherein the bone marrow iscultured for about 3 days in the hypoxic environment.
 33. The method ofclaim 25, wherein the hypoxic-activated marrow infiltrating lymphocytesare cultured for about 6 days in the normoxic environment.
 34. Themethod of claim 25, wherein the hypoxic-activated marrow infiltratinglymphocytes are cultured for about 9 days in the normoxic environment.35. The method of claim 25, wherein the cancer is a hematologicalcancer.
 36. The method of claim 25, wherein the cancer is lung cancer.37. The method of claim 25, wherein the cancer is breast cancer.
 38. Themethod of claim 25, wherein said normoxic environment comprises betweenabout 21% oxygen and about 30% oxygen.
 39. The method of claim 25,wherein said normoxic environment comprises between about 21% oxygen andabout 25% oxygen.
 40. The method of claim 25, wherein said normoxicenvironment comprises between about 21% oxygen and about 24% oxygen. 41.The method of claim 25, wherein said normoxic environment comprisesbetween about 21% oxygen and about 23% oxygen.